欢迎访问作物学报，今天是
作物学报

作物学报 ›› 2010, Vol. 36 ›› Issue (05): 788-793.doi: 10.3724/SP.J.1006.2010.00788

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

根癌农杆菌介导的转双价抗虫基因(CryIA+CpTI)苎麻

马雄风1,3,喻春明1,唐守伟1,郭三堆2,张锐2,王延周1,朱爱国1,朱四元1,熊和平1,*   

  1. 1中国农业科学院麻类研究所/农业部麻类遗传改良与工程微生物重点开放实验室,湖南长沙410205;2中国农业科学院生物技术研究所,北京100081;3中国农业科学院棉花研究所,河南安阳455000
  • 收稿日期:2009-08-26 修回日期:2009-12-21 出版日期:2010-05-12 网络出版日期:2010-03-15
  • 通讯作者: 熊和平,E-mail:ramiexhp@2118.cn;Tel:0731-88998500
  • 基金资助:

    本研究由国家"十一五"科技支撑计划项目(2006BAD06B03)和国家公益性行业(农业)科研专项项目(nyhyzx07-018)资助.

Transgenic of Ramie with Synthetic CryIA+CpTI Gene by Agrobacterium tumefaciens-Mediated

 MA Xiong-Feng1,3,YU Chun-Meng1,TANG Shou-Wei1,GUO San-Dui2,ZHANG Dui2,WANG Yan-Zhou1,SHU Ai-Guo1,SHU Si-Yuan1,XIONG He-Beng1*   

  1. 1Institute of Bast Fiber Crops,Chinese Academy of Agricultural Sciences/Key Laboratory of Genetic Improvement & Engineering Microbiology for Bast Fiber Crops,Changsha 420105,China;2Biotechnology Research Center,Chinese Academy of Agricultural Sciences,Beijing 100081 China;3Institute of Cotton Research,Chinese Academy of Agricultural Sciences,Anyang 455000,China
  • Received:2009-08-26 Revised:2009-12-21 Published:2010-05-12 Published online:2010-03-15
  • Contact: XIONG He-Ping,E-mail:ramiexhp@2118.cn;Tel:0731-88998500

PDF

1326

被引次数

9

摘要：

利用苎麻下胚轴高频再生体系，将携带人工合成的CryIA杀虫基因和CpTI基因的高效双价杀虫基因的植物表达载体pGBI4ABC转化到苎麻主栽品种中苎1号中。经根癌农杆菌侵染和共培养后，用50 mg L–1卡那霉素+ 300 mg L–1头孢霉素筛选共获得32株抗性植株; PCR检测显示26株为阳性, 占80%。Southern杂交结果证实, 外源基因已经整合到苎麻的基因组中。这项研究为最终创造兼抗鳞翅目及鞘翅目等害虫的苎麻种质材料奠定了基础。

关键词: 苎麻, 抗虫基因, 根癌农杆菌, 转基因植株

Abstract:

Ramie [Boehmeria nivea L. (Guad.)] is a perennial herbaceous plant of the urticaceae family. Ramie affected by many insect pests above and below the ground during the growing season. The pests damage has become an important factor affecting ramie yield and quality in China. Application of chemical insecticides not only is less effective and costly, but also causes environmental pollution. Development of a genetic transformation system to introduce desired genes (e.g. toxin genes) into ramie could improve its agromonic traits such as resistance to insects. The study was conducted to transfer CryIA gene and CpTI gene into the ramie variety Zhongzhu 1. The CryIA gene encodes Bt toxin protein from Bacillus thuringiensis toxinof Vitis pseudoreticulata; while, the CpTI gene encodes a cowpea trypsin inhibitor produced by Vigna unguiculata. Synthesized CryIA geneand CpTI gene were inserted into plant expression vectorpGBI4ABC whichcarried high efficient bivalent insect resistant genes. With high frequency of adventitious shoot induction and plant regeneration, ramie hypocotyls of the variety Zhongzhu 1 were transformed with Agrobacterium tumefaciens strain LB4404 harboring the plant expression vector pGBI4ABC, and then shoot buds formed from calli induced from hypocotyls were selected with the mixed solution of 50 mg L–1 Kanamycin and 300 mg L–1 Cefotaxime. The result showed that 32 Kanamycin-resistant plants were obtained, and therein 26 plants were positive in PCR test, indicating that 80% of the Kanamycin-resistant plants contain CryIA gene and CpTI gene. Southern blot and GUS analysis also verified that the foreign genes of CryIA and CpTI were successfully integrated into the genome of the transgenic plants and expressed.

Key words: Ramie, Insect resistant genes, Agrobacterium tumefaciens, Transegenetic plants

[1] Xiong H-P(熊和平). Breeding of Plant Fiber Crops (麻类育种学). Beijing: China Agricultural Science and Technology Press, 2008

[2] Li F-G(李付广), Cui J-J(崔金杰), Liu C-L(刘传亮), Wu Z-X(武芝霞), Li F-L(李凤莲). 中国农业科学), 2000, 33(1): 46–52 (in Chinese with English abstract)The study of insect-resistant transgenic cotton harboring double-gene and its insect-resistance. Sci Agric Sin (

[3] Cui H-Z(崔洪志), Guo S-D(郭三堆). Construction of plant expression vectors harboring two insecticidal genes and their expression in tobacco. J Agric Biotechnol (农业生物技术学报), 1998, 6(1): 7–13 (in Chinese with English abstract)

[4] Joseph S, Rusell D W. Molecular Cloning: A Laboratory Manual (分子克隆实验指南).Beijing: Science Press, 2002, pp 487–510 (in Chinese)

[5] Huang X-Y(黄小英), Liu Y(刘瑛), Lai X-P(赖小萍). A DNA extract method for wild ramie. Chin Wild Plant Resour (中国野生植物资源), 2002, 22(1): 1–5 (in Chinese)

[6] Jefferson J, Kavanagh T, Bevan M. GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J, 1987, 6: 3901–3907

[7] Wang G-L(王关林), Fang H-J(方宏筠). Plant Genetic Engineering (植物基因工程). Beijing: Science Press, 2002. pp 344, 361, 385–388

[8] Dusi D M A, Dubald M, De Almeida R P, Caldas L S, Gander E S. Transgenic plants of ramie (Boehmeria nivea Gaud.) obtained by Agrobacterium mediated transformation. Plant Cell Rep, 1993, 12: 625–628

[9] Yi Z-L(易自力), Li X(李祥), Jiang J-X(蒋建雄), Wang Z-C(王志成), Liu Q-B(刘清波). The establishment of regeneration system and obtaining the insect-resistant transgenic plants of ramie (Boehmeria nivea L.). China Fiber Crops (中国麻业), 2006, 28(2): 61–66 (in Chinese)

[10] Wang B(汪波), Peng D-X(彭定祥), Sun Z-X(孙珍夏), Zhang N(张娜), Xing X-L(邢秀龙). Regeneration of transgenic ramie plants expressing green fluorescent protein mediated by Agrobacterium tumefaciens. Acta Agron Sin (作物学报), 2007, 33(10): 1606–1610 (in Chinese)

[11] Wang B, Peng D X, Sun Z X, Zhang N, Gao S M. In vitro plant regeneration from seedling-derived explants of ramie

[Boehmeria nivea L. (Gaud.)]. In Vitro Cell & Develop Biol-Plant, 2008, 47: 105–111

[12] Wang B, Peng D X, Liu L J, Sun Z X, Zhang N, Gao S M. An efficient adventitious shoot regeneration system for ramie (Boehmeria nivea Gaud.) using thidiazuron. Bot Studies, 2007, 48: 173–180

[13] Ma X-F(马雄风), Yu C-M(喻春明), Xiong H-P(熊和平). Progress of tissue culture and genetic transformation in ramie. Mol Plant Breed (分子植物育种), 2008, 6(5): 967–970 (in Chinese)

[14] Chen D-F(陈德福), Chen X-W(陈喜文). Preliminary study on somatic embryogenesis of ramie. Chin Bull Bot (植物学通报), 1998, 15(3): 65–68 (in Chinese)

[15] Huang J-S(黄记生), Mo R-D(莫荣达). In vitro organogenesis of ramie Boechmeria nivea (L.) Gaud. J Mol Cell Biol (分子细胞生物学报), 1981, 14(2): 111–114 (in Chinese)

[16] Mo R-D(莫荣达), Huang J-S(黄记生). The effects of hormone on the differentiation of stem and leaf explants of ramie. Plant Physiol Commun (植物生理学通讯), 1983, (3): 39–41 (in Chinese)

[17] Zhao Q-H(赵庆华), Yan C-J(颜昌敬), Pan C-L(潘昌立), Yang M-Q(杨泌泉), Wang C-T(王春桃), Li Z-D(李宗道). Studies on tissue culture of leaves from ramie. China Fiber Crops (中国麻作), 1984, (4): 11–13 (in Chinese)

[18] Ma X F, Yu C M, Xiong H P. Genetic transformation of the bast fiber plant ramie (Boehmeria nivea Gaud.) by Agrobacterium tumefaciens-mediated method. Plant Cell, Tissue and Organ Culture, 2010, 100: 165–174




[19] James C. Global status of commercialized biotech/GM Crops: 2007. ISAAA Briefs No 37, International Service for the Acquisition of Agri-Biotech Applications, Ithaca, NY, 2007
[1] 李富, 王延周, 严理, 朱四元, 刘头明. 苎麻茎皮环状RNA表达谱分析[J]. 作物学报, 2021, 47(6): 1020-1030.
[2] 付虹雨, 崔国贤, 李绪孟, 佘玮, 崔丹丹, 赵亮, 苏小惠, 王继龙, 曹晓兰, 刘婕仪, 刘皖慧, 王昕惠. 基于无人机遥感图像的苎麻产量估测研究[J]. 作物学报, 2020, 46(9): 1448-1455.
[3] 陈倩楠,王轲,汤沙,杜丽璞,智慧,贾冠清,赵宝华,叶兴国,刁现民. 以抗除草剂Bar基因稳定转化谷子技术研究[J]. 作物学报, 2018, 44(10): 1423-1432.
[4] 郝岭,张钰石,段留生,张明才*,李召虎. 玉米ZmBRI1基因的克隆、表达及功能分析[J]. 作物学报, 2017, 43(09): 1261-1271.
[5] 唐映红, 陈建荣, 刘芳, 袁有美, 郭清泉, 昌洪涛. 苎麻肉桂酰辅酶A还原酶基因cDNA序列的克隆与分析[J]. 作物学报, 2015, 41(09): 1324-1332.
[6] 刘昱翔,陈建荣,彭彦,黄妤,赵燕,黄丽华,郭清泉,张学文. 两种苎麻纤维素合酶基因cDNA序列的克隆及表达[J]. 作物学报, 2014, 40(11): 1925-1935.
[7] 周精华，余伟林，邢虎成,揭雨成，钟英丽，敬礼恒. 苎麻ACC合酶基因(BnACS1)的克隆和表达分析[J]. 作物学报, 2012, 38(12): 2306-2311.
[8] 邹自征，陈建华，栾明宝，郭劲霞，王超，王晓飞，许英，孙志民. 应用RSAP、SRAP和SSR分析苎麻种质亲缘关系[J]. 作物学报, 2012, 38(05): 840-847.
[9] 周精华, 邢虎成, 揭雨成, 钟英丽, 朱守晶, 蒋杰, 王亮. 苎麻Δ1-吡咯啉-5-羧酸合成酶(P5CS)基因的克隆和表达分析[J]. 作物学报, 2012, 38(03): 549-555.
[10] 佘玮, 揭雨成, 邢虎成, 鲁雁伟, 黄明, 康万利, 王栋. 苎麻耐镉品种差异及其筛选指标分析[J]. 作物学报, 2011, 37(02): 348-354.
[11] 栾明宝, 陈建华, 许英, 王晓飞, 孙志民. 苎麻核心种质构建方法[J]. 作物学报, 2010, 36(12): 2099-2106.
[12] 黄天带,李哲,孙爱花,周权男,华玉伟,黄华孙. 根癌农杆菌介导的橡胶树花药愈伤组织遗传转化体系的建立[J]. 作物学报, 2010, 36(10): 1691-1697.
[13] 马雄风,喻春明,唐守伟,朱爱国,王延周,朱四元,刘建新,熊和平. 苎麻Actinl基因克隆及其在韧皮部纤维不同发育阶段的表达[J]. 作物学报, 2010, 36(1): 101-108.
[14] 符家平，汪波，刘立军，杨金雨，王绪霞，邢秀龙，彭定祥. 根癌农杆菌介导转Bt基因苎麻的获得及其抗虫鉴定[J]. 作物学报, 2009, 35(10): 1771-1777.
[15] 廖亮，李同建，刘中来，邓辉胜，徐玲玲，潘其辉，赖占均，石庆华. 基于细胞学和DNA序列的苎麻与其野生近缘类群系统关系研究[J]. 作物学报, 2009, 35(10): 1778-1790.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备15008789号-2